Device and method for combined microfluidic-micromagnetic separation of material in continuous flow

ABSTRACT

A miniaturized, integrated, microfluidic device pulls materials bound to magnetic particles from one laminar flow path to another by applying a local magnetic field gradient. The device removes microbial and mammalian cells from flowing biological fluids without any wash steps. A microfabricated high-gradient magnetic field concentrator (HGMC) is integrated at one side of a microfluidic channel. When magnetic particles are introduced into one flow path, they remain limited to that flow path. When the HGMC is magnetized, the magnetic beads are pulled from the initial flow path into the collection stream, thereby cleansing the fluid. The microdevice allows large numbers of beads and materials to be sorted simultaneously, has no capacity limit, does not lose separation efficiency as particles are removed, and is useful for cell separations from blood and other biological fluids. This on-chip separator allows cell separations to be performed in the field outside of hospitals and laboratories.

FIELD OF THE INVENTION

The present invention relates to systems and methods for separatingmaterials from biological fluids. More particularly, it relates todevices and methods for quickly and efficiently separating cells ormolecules from biological fluids in a microfluidic channel using ahigh-gradient magnetic concentrator.

BACKGROUND OF THE INVENTION

One of the key functions required for microsystems technologies used forbiomedical applications is to separate specific cells or molecules fromcomplex biological mixtures, such as blood, urine, or cerebrospinalfluid. For example, hemofiltration and hemadsorption techniques removeimpurities or pathogens in blood. Various physical properties, includingsize (Huang et al., 2004; Yamada et al., 2004), motility (Cho et al.,2003), electric charge (Lu et al., 2004), electric dipole moment(Fiedler et al., 1998; Hunt et al., 2004), and optical qualities (Fu etal., 1999; Wang et al., 2005), have been studied to separate specificcells or molecules from these mixtures. Magnetic susceptibility also hasbeen explored (Pamme, 2006) because magnetic sorting can be carried outat high-throughput in numerous biological fluids with minimal powerrequirements, and without damaging the sorted entities (Franzreb et al.,2006; Hirschbein et al., 1982; Lee et al., 2004; Safarik and Safarikova,1999; Setchell, 1985). Biocompatible superparamagnetic particles arealso now available with surfaces modified to promote binding to variousmolecules and cells. In fact, various macroscale magnetic sortingsystems have been built and employed for research and clinicalapplications (Chalmers et al., 1998; Fuh and Chen, 1998; Handgretingeret al., 1998; Hartig et al., 1995; Melville et al., 1975a; Takayasu etal., 2000) (e.g., to isolate stem cells from batches of pooled blood forbone marrow reconstitution procedures in cancer patients (Handgretingeret al., 1998)).

Batch-type magnetic separators have been microfabricated on single chipsthat trap magnetic particles in flowing fluids using an externalmagnetic field, and then the particles are later eluted from the system(Ahn et al., 1996; Deng et al., 2002; Smistrup et al., 2005; Tibbe etal., 2002). However, the loading capacity of these devices is limitedbecause accumulation of the collected particles can restrict fluid flowor lead to irreversible entrapment of samples, and the use of thesesystems is hampered by the need to disrupt continuous operation forsample elution.

Further, continuous on-chip separation may simplify microsystemoperation and potentially improve separation efficiency. In particular,microfluidic systems that are extensively utilized in micro-totalanalysis systems (μTAS) offer the potential to separate componentscontinuously from flowing liquids. Continuous separation of magneticparticles in microfluidic channels has been demonstrated by manuallyplacing a permanent magnet or electromagnet beside a microchannel thatcontains multiple outlets (Blankenstein, 1997; Kim and Park, 2005; Pammeand Manz, 2004). However, because each magnet needs to be individuallyfabricated and positioned, further miniaturization and multiplexing isnot possible with this approach.

Additionally, high-gradient magnetic concentrators (HGMCs) can generatea large magnetic force with simple device structures. Macroscale HGMCshave been used in magnetic separations for biomedical applications(Chalmers et al., 1998; Fuh and Chen, 1998; Hartig et al., 1995;Melville et al., 1975a; Takayasu, 2000), but are impractical formicrosystems technologies due to their large dimensions. With thedevelopment of microfabrication technologies, it has become possible tomicrofabricate HGMCs along with microfluidic channels on a single chip.Several on-chip HGMC-microfluidic designs for continuous magneticseparation have been reported (Berger et al., 2001; Han and Frazier,2004, 2006; Inglis et al., 2004). One design used microfabricatedmagnetic stripes aligned on the bottom of the fluid chamber tohorizontally separate magnetically tagged leukocytes trapped on themagnetic stripes away from red blood cells (RBCs) flowing through thechamber (Inglis et al., 2004). In another design, a microfabricatedmagnetic wire was placed in the middle of the flow stream along thelength of a single microfluidic channel, and used to separatedeoxyhemoglobin RBCs from white blood cells based on the difference intheir relative magnetic susceptibilities (Han and Frazier, 2006).

However, none of these designs provides for portable devices forin-field diagnosis or treatment of diseases caused by blood-bornepathogens, such as sepsis—the body's systemic response to infection inthe blood. The overall death rate from this blood infection is 25% inthe United States and higher internationally, and in the military fieldof operation. In a septic patient, the blood becomes overloaded with arapidly growing infectious agent, and other clearance mechanisms areovercome. The prior attempts have fallen short in that no devicepresently exists that can rapidly clear infectious pathogens from bloodand biological fluids without causing significant blood loss,obstructing blood flow, otherwise altering blood content, orcompromising normal organ function.

Thus, there is a need for a device that can rapidly cleanse the blood ofpathogens and other biological particulate materials without removingcritical normal blood cells, proteins, fluids, or electrolytes. Also,there is a need for biocompatible magnetic labeling particles that willselectively bind to living pathogens within flowing blood such asnatural opsonins, while being small enough so that they will not producevascular occlusion. Moreover, there is a need for a microfluidicseparator generating appropriate magnetic field gradients in amicrofluidic device for continuous separation of materials frombiological fluids in biomedical and biophysical diagnosis and treatmentapplications.

SUMMARY OF THE INVENTION

The present invention provides technologies for magnetic separation ofliving cells from biological fluids such as blood, cerebrospinal fluid,urine, and the like. The present invention can be used in portabledevices for in-field diagnosis or therapy of diseases caused byblood-borne and other pathogens, such as sepsis and the like. The deviceand method of the present invention may be employed as on-chip magneticseparation and be used for isolating rare cells, such as cancer cells,stem cells, or fetal cells in the maternal circulation, and the like.The present invention incorporates nanoscale magnetic particles intobiological fluids in continuous flow. The biocompatible magneticnanoparticles bind pathogenic or other cells in the fluid and may becleared from the fluid by applying an in-line high gradient magneticconcentrator (HGMC) to move magnetically susceptible material across alaminar streamline boundary into a collection stream flow path. Thepresent invention provides an on-chip HGMC-microfluidic system anddevice that offers improvements over existing designs in terms ofbiocompatibility, separation efficiency, and rate of clearance, whileminimizing the disturbance on normal blood cells and biomolecules. Themicrofabricated on-chip HGMC-microfluidic system of the presentinvention efficiently separates magnetic micro- and nano-particles,either alone or bound to living cells, under continuous fluid flow.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate an embodiment of the invention anddepict the above-mentioned and other features of this invention and themanner of attaining them. In the drawings:

FIG. 1 shows a schematic illustration of an on-chip HGMC-microfluidicseparator in accordance with an embodiment of the present invention.

FIGS. 2A-2B show a schematic illustration of an on-chipHGMC-microfluidic separator with a detailed view of the microfluidicchannel in accordance with an embodiment of the present invention.

FIGS. 3A-3E illustrate bright field microscopic images comparing theflow pattern of magnetic beads in a microfluidic channel in the absenceand presence of a microfabricated high gradient magnetic fieldconcentrator and graphical depictions of the corresponding magneticfield, magnetic field gradient, and magnetic field distribution patternin accordance with the present invention.

FIGS. 4A-4C illustrate a microscopic view of a high gradient magneticfield concentrator, the corresponding magnetic field and magnetic fieldgradient, and a simulated magnetic field distribution in accordance withan embodiment of the present invention.

FIGS. 5A-5C illustrate magnetic separations of magnetic and non-magneticbeads cells using a combined microfluidic-micromagnetic separator inaccordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description of the invention refers to theaccompanying drawings and to certain embodiments, but the detaileddescription of the invention does not limit the invention. The scope ofthe invention is defined by the appended claims and equivalents as itwill be apparent to those of skill in the art that various features,variations, and modifications can be included or excluded based upon therequirements of a particular use.

The present invention extends the functionality of current microfluidicsystems techniques to provide on-chip technologies for magneticseparation of living cells from biological fluids such as blood,cerebrospinal fluid, urine, and the like. The present invention combinesmagnetic and microfluidic separation technologies in continuous flowapplications thereby creating effectively unlimited binding andclearance capacity that can be used in portable devices for in-fielddiagnosis or therapy of diseases, for example, diseases caused byblood-borne pathogens, such as sepsis and the like. The system andmethod of the present invention has many advantages over prior systemssince the micromagnetic-microfluidic separation techniques of thepresent invention may be multiplexed to offer high throughput fielddiagnosis and treatment options. The separation technologies may also beused in an on-chip environment for isolating rare cells, such as cancercells, stem cells, and the like. Further, the present invention offersimprovements over existing designs in terms of biocompatibility,separation efficiency, and rate of clearance, while minimizing thedisturbance on normal blood cells and biomolecules. The microfabricatedon-chip high gradient magnetic field concentrator (HGMC)-microfluidicsystem of the present invention efficiently separates magnetic micro-and nano-particles, either alone or bound to living cells, such asbacteria, under continuous fluid flow.

Separation Microsystem Overview

As shown schematically in FIG. 1, an embodiment of the separationmicrosystem 100 of the present invention incorporates magnetic particles101 that may be injected into the fluid 150. Magnetic particles 101 bindto material (not shown separately) in the fluid 150. Fluid 150 entersmicrofluidic channel 110 through source inlet 160 and flows throughmicrofluidic channel 110. Microfluidic channel 110 includes at least onedimension less than 1 mm and exhibits laminar flow. The fluid 150 mayflow in discrete volumes or may flow continuously to increase efficiencyin the separation techniques of the present invention.

Microfluidic channel 110 includes a streamline boundary 120 thateffectively separates the source flow path 122 from the collectionstream flow path 124 in the microfluidic channel 110. A high gradientmagnetic field concentrator 190 is magnetized to move the material boundto magnetic particles 101 to cross the streamline boundary 120 and enterthe collection stream flow path 124 to effectively and efficientlyseparate the material bound to magnetic particles 101 from the fluid 150in the microfluidic channel 110.

As fluid 150 flows in the microfluidic channel 110, the material boundto magnetic particles 101 and affected by the high gradient magneticfield concentrator 190 flows in the collection stream flow path 124 to acollection outlet 180. The fluid 150 and any other material not affectedby the magnetized high gradient magnetic field concentrator 190 does notcross the streamline boundary 120 and thereby remains in the source flowpath 122.

As fluid 150 flows in the microfluidic channel 110, material unaffectedby the magnetized high gradient magnetic field concentrator 190continues to flow in the source flow path 122 and exits the microfluidicchannel 110 through source outlet 170. Separation microsystem 100 can beused to continuously transfer material labeled with magnetic particles101 out of the source flow path 122 to a collection outlet 180 toprovide high throughput cleansing of fluid 150.

Separation Microsystem Fabrication

In another embodiment, the microfluidic channel 110 may be prepared bysoft lithography (McDonald and Whitesides, 2002) and have dimensions of20×0.2×0.05 mm (L×W×H), for example. A negative mold of the microfluidicchannel 110 may be produced in SU-8 photoresist (Microchem, Inc.).Poly(dimethylsiloxane) (PDMS) (Slygard 184, Dow Corning) may be pouredonto the mold, allowed to cure for 1 hour at 65° C., and peeled off toform the microfluidic channel 110. A lift-off process (Wolf, 1986) maybe used to microfabricate the high gradient magnetic field concentrator190 by defining a base layer of evaporated metal (Ti/Au, 10 nm/50 nm) inthe form of a microneedle (20 mm in X, 100 μm in Y, 50 μm in Z) or amicrocomb (3.8 mm in X, 12 mm in Y, 50 μm in Z with teeth 300 μm in Xand spaced by 200 μm in Y), or a magnetic line array, for example, on aglass substrate that may then be electroplated (1 mA for 4 hr) with a 50μm thick layer of magnetic material (80% Ni, 20% Fe) (Rasmussen et al.,2001). The geometry of the high gradient magnetic field concentrator 190is used to produce a steep, uniform magnetic field gradient across thewidth of the microfluidic channel 110. Electrodeposition andphotolithography may be used to fabricate a high gradient magnetic fieldconcentrator of magnetic material such as NiFe or the like. In oneembodiment of the present invention, a microfluidic channel wasfabricated containing a 12 mm long layer of NiFe placed along a sideedge of the microfluidic channel as a soft magnetic high gradientmagnetic field concentrator. The PDMS microfluidic channel 110 and theglass substrate with the NiFe layer may then be exposed to oxygen plasma(100 W, 60 sec) and bonded together to form a separation microsystem 100of the present invention.

A soft magnetic material, such as NiFe, for example, with low remnantmagnetization was magnetized with an external stationary magnet tofacilitate rapid and switchable control of cell separations. The systemof the present invention may also be microfabricated using permanentmagnetic materials or incorporate elements that provide electromagneticcontrol. Moreover, the same microfabrication techniques could be used todeposit multiple magnetic layers at different positions on one chipsimultaneously, thus providing multiplexing capabilities in accordancewith the present invention.

Magnetic Particles (Beads and Cells) Used to Label Target Material

In one embodiment of the present invention, non-magnetic red-fluorescentbeads (2 μm diameter, 4.5×10⁹ beads/ml, Molecular Probes) andsuperparamagnetic green-fluorescent beads (1.6 μm, 43% iron oxide,3.1×10⁹ beads/ml, Bangs Laboratories) were incubated in 10× volume of 1%albumin solution for 1 hour before being combined and injected into themicrofluidic channel 110. E. coli (HB101 K-12) bacteria expressing greenfluorescent protein (GFP) were grown overnight at 37° C. in LB mediumcontaining ampicillin (100 μg/ml) and arabinose (0.1%, inductor of GFPexpression), then harvested and resuspended in PBS buffer. The E. coli(1×10⁹ CFU/ml) were labeled with biotinylated anti-E. coli antibody(Virostat; mixing ratio 2 μg antibody/10⁷ cells), and mixed withstreptavidin-coated superparamagnetic particles (130 nm, 85% iron oxide,G. Kisker GbR) prior to addition to the microfluidic separation system100. Human RBCs (75% hematocrit) were obtained from the blood bank atChildren's Hospital Boston, stained with the red fluorescent dye (SYTO64, Molecular Probes), and mixed with isotonic saline containing 0.5%albumin at a 1:3 ratio (final density around 2×10⁹ RBCs/ml).

Microfluidic Control Testing

Fluidic connections, such as source inlet 160, source outlet 170,collection inlet 162, and collection outlet 180, to the microfluidicchannel 110 were made with polyethylene tubing inserted through holespunched through the PDMS. Syringe pumps were used to control the flowrate at each of the inlets 160, 162 independently. Prior to each test,the microfluidic channel 110 and tubing were cleaned by flushing with70% ethanol, rinsing with deionized water, and incubating in phosphatebuffered saline (PBS) with 1% albumin for 30 min. The fluid 150containing the sample target material and a dextran solution (32%, 70kDa) were injected simultaneously into the source and collection inlets160, 162, respectively. All tests were carried out using experimentalsamples contained within the first half of the volume from syringes inthe upright position. Separations of target materials and the magneticparticles in the microfluidic channel 110 were monitored in real-timeusing an inverted Nikon TE2000-E microscope equipped with a CCD camera,and optimized by adjusting the fluid flow rate and the output splitratio. The width of the source flow path was maintained as ⅓-½ of themicrofluidic channel width.

While some cell separation techniques such as fluorescence-activatedcell sorting (FACS) have achieved a sorting rate of approximately 100cells/s (Wang et al., 2005), because FACS and similar systems are serialprocesses allowing only one cell to pass through the actuator at a time,further increases in sample throughput require improvement in the cycletime of the actuator. In contrast, the throughput of the micromagneticseparator of the present invention increases when the cell density ofthe sample is raised, and a cell throughput of 10,000 cells/s isdemonstrated. This enhanced throughput is possible because the widesource path used here (⅓-½ of channel width) allows large numbers ofbeads and cells to pass through the separating magnetic field gradientsimultaneously. Thus, the system and device of the present invention isespecially useful for separations from blood or other clinical sampleswith high cell density and low optical transparency.

In addition to increasing the width of the channel flow path, adisk-shaped (4 mm diameter, 2 mm high, magnetized along the z-axis)neodymium permanent magnet was used to magnetize the NiFe layer highgradient magnetic field concentrator 190. Of course, any suitable magnetsource may be used to magnetize the gradient magnetic field concentrator190, including for example, permanent magnets, electromagnets,paramagnetic sources, and the like. Likewise, the magnetic source may beinherent to the high gradient magnetic field concentrator, such as whenthe high gradient magnetic field concentrator includes permanentmagnetic materials that do not require an external magnetizing field. Inone embodiment of the present invention, the neodymium permanent magnetwas positioned in the middle of the NiFe layer in the flow directionwith its center 4 to 5 mm from the closest side of the microfluidicchannel 110 using a microscope micromanipulator. A schematicconfiguration of the separation microsystem 200 is shown also in FIG. 2Aand FIG. 2B with a detailed view of the microfluidic channel.

Quantification of Separation Efficiencies

Quantification of clearance efficiency using the fluorescent microbeadsmay be performed using the inverted Nikon TE2000-E microscope or anysimilar examination device by measuring the fluorescence intensity ofthe collected fluids from both outlets 170, 180. In one embodiment ofthe present invention using E. coli, the high density of bound magneticnanoparticles blocked the green fluorescent protein (GFP) signal. Thus,collected bacterial numbers may be quantified by transferring the fluidscollected from the outlets 170, 180 to a growth medium and culturing at37° C. The optical density of the cell solutions at 600 nm (OD_(600nm))may be measured periodically. Cell numbers may be determined usingOD_(600nm) obtained during the logarithmic phase of growth, andOD_(600nm) during this phase was linearly related to the startingconcentration of the magnetically-labeled E. coli bacteria.

Operation of the Separation Microsystem

FIG. 2A shows a three dimensional schematic view of an on-chipHGMC-microfluidic separator 200 with a detailed inset view of themicrofluidic channel in accordance with an embodiment of the presentinvention. The microfabricated on-chip microfluidic-micromagnetic cellseparator of the present invention may be used to continuously cleansecontaminant materials such as bacteria, particulates, and the like frombiological fluids. FIG. 2B illustrates a cross sectional view of themicrofluidic separator. Where possible in FIG. 2A and FIG. 2B, likereference numerals have been used to designate like features that arecommon to FIG. 1.

As shown schematically in FIG. 2A, the combinedmicromagnetic-microfluidic separation system 200 contains a highgradient magnetic concentrator 290 adjacent to the microfluidic channel210. The high gradient magnetic concentrator 290 may contain amicrofabricated layer of soft magnetic material, such as NiFe, or othersuitable magnetic materials. The single microfluidic channel 210 isconnected to two inlets 260, 262 and two outlets 270, 280. Due to thesmall Reynolds number (Re) of microfluidic channels, the flow of fluid250 remains laminar with mixing due only to diffusion across the sourceflow path 222 and the collection stream flow path 224. In one embodimentof the present invention, a layer of magnetic material such as NiFe withthe same thickness as the height of the microfluidic channel 210 may bedeposited adjacent to the channel 210 during the microfabricationprocess to create an on-chip high gradient magnetic concentrator 290(HGMC) with defined geometry such as a microneedle, microcomb, magneticline array, and the like.

The thickness of the high gradient magnetic concentrator 290 correspondsto the height of the microfluidic channel 210 to ensure that themagnetic particles 201 and target materials flowing at different heightsthrough the microfluidic channel 210 were exposed to similar magneticfield gradients. While channel height does not affect the separationefficiency of the system of the present invention, it influences thevolume flow rate. In one embodiment of the present invention, arelatively small channel height (50 μm) was chosen to facilitatereal-time focusing and monitoring of flows in the channel undermicroscopic visualization. Higher volume throughput may be achieved byincreasing the channel height and the magnetic layer thickness inparallel. As an example, a fluidic channel that is centimeters long witha source flow path and a collection stream flow path each 100 micronswide may be employed, but instead of a channel height of 50 microns, thechannel height may be centimeters high. The sorting rate will scaleupward with the height of the channel.

When magnetized by an external magnet, the HGMC 290 locally concentratesthe gradient of the applied magnetic field to move the magneticparticles 101 that are present in the source flow path 222 (shown indetail as upper path 222 in the FIG. 2A inset) across the laminar flowstreamline boundary 220 and into the neighboring collection stream flowpath 224 (also shown in detail as lower path 224 in the FIG. 2A inset).The external magnet may be any suitable magnet, such as a permanentmagnet, an electromagnet, or the like. In one embodiment of the presentinvention, an external permanent neodymium magnet was used to magnetizethe HGMC 290 of the present invention.

FIG. 2A inset further illustrates how magnetic particles 201, such asmagnetic beads, flowing in the upper source flow path 222 are pulledacross the laminar streamline boundary 220 into the lower collectionstream flow path 224 when subjected to a magnetic field gradientproduced by the microfabricated NiFe layer located along the lower sideof the microfluidic channel 210. Also shown in the FIG. 2A inset, fluidflow is illustrated in the y-direction, the magnetic field gradientacross the channel is in the x-direction, and the channel height is inthe z-direction.

The affected particles 201 will then exit through the lower collectionoutlet 280. Under the same conditions, non-magnetic particles 202 in thesource flow path are unaffected by the applied magnetic field gradient,and thus, they will exit the microfluidic channel 210 through the uppersource outlet 270.

FIG. 3A and FIG. 3B were constructed by overlaying sequential frames oftime-lapse movies recorded at the middle of the microfluidic channel ofthe present invention. FIG. 3A illustrates the flow pattern of magneticbeads in the absence of a high gradient magnetic field concentrator,while FIG. 3B shows the flow pattern of magnetic beads in the presenceof a high gradient magnetic field concentrator in accordance with thepresent invention.

As shown in FIG. 3A, in one embodiment of the present invention, avolume flow rate of 5 μl/hr (0.3 mm/s) was established through themicrofluidic channel. At this flow rate, the microfabricated separationdevice containing a magnetized NiFe high gradient magnetic fieldconcentrator 290 in the form of a microneedle oriented perpendicularlyto the flow paths 222, 224 and juxtaposed to the side of themicrofluidic channel 210 drove the magnetic particles 201 (magneticbeads with 1.6 μm diameter) flowing in the upper source flow path 222 tocross over the streamline boundary 220 and enter the lower collectionstream flow path 224 as depicted in FIG. 3B. The magnetic particles 201eventually exited the microfluidic channel 210 through the collectionoutlet 280. This separation was possible because the NiFe high gradientmagnetic field concentrator 290 microneedle generated a strongermagnetic field gradient across the channel (i.e., greater than 25 T/m),with a field strength in the vertical and horizontal directions ofgreater than 0.016 and greater than 0.013 T, respectively, whenmagnetized by the magnetic source of the external neodymium magnet.Additional magnetic sources such as paramagnetic sources,electromagnets, and the like, may also be used to magnetize the highgradient magnetic field concentrator 290. Additionally, the magneticsource may be inherent to the high gradient magnetic field concentrator,such as when the high gradient magnetic field concentrator includespermanent magnetic materials that do not require an external magnetizingsource. The results of the magnetization using the neodymium magnet areshown in FIG. 3D and FIG. 3E, and are described further in the Appendix.The corresponding magnetic field is illustrated as a function ofdistance from the collection stream flow path channel wall 297 in FIG.3C, and in FIG. 3D, the magnetic field gradient is presented as afunction of distance from the collection stream flow path channel wall297. The solid, dashed, and dotted lines in the graphs of FIG. 3C andFIG. 3D correspond to the vertical magnetic field (B_(z)), horizontalmagnetic field (B_(x)) and the magnetic field gradient across thechannel

$\left( {\frac{\overset{\rightarrow}{B}}{x} \cdot \hat{B}} \right),$

respectively. In FIG. 3E, computer-simulated magnetic fielddistributions are depicted as grayscale variations within themicrofluidic channel generated by the magnetized high gradient magneticfield concentrator 290 in the form of a NiFe microneedle. Both top (topFIG. 3E) and cross-sectional (bottom FIG. 3E) views are illustrated.

Additionally, to increase throughput, volume flow rates may be increasedto 25 μl/hr. With increased volume flow rates, stronger magnetic fieldgradients need to be produced by the high gradient magnetic fieldconcentrator 290 to maintain the separation efficiency of themicrofluidic separation device 200.

For example, to increase the separation efficiency of the on-chipHGMC-microfluidic separator, one embodiment of the present inventionemploys a microfabricated NiFe layer in a microcomb configuration thathas a triangular saw-tooth edge as shown in FIG. 4A. In operation, thetriangular saw-tooth edged high gradient magnetic field concentrator ispositioned close to the side of the microfluidic channel. Due to itshigh curvature geometry, the microcomb high gradient magnetic fieldconcentrator concentrates the magnetic field and produces a steepmagnetic field gradient across the width of the microfluidic channel 210without providing excessive trapping of particles 201 near the channelwall. The saw-tooth edge of the microcomb provides horizontal andvertical magnetic fields of 0.025 T and 0.018 T, respectively, at thefar edge of the channel, and a field gradient of 50 T/m as shown in FIG.4B and FIG. 4C and described further in the Appendix. Additionalsuccessful tests were performed with a magnetic field gradient over arange of 1 to 500 T/m, with preferred results in the range of 15 to 150T/m. In addition, the region of the microfluidic channel exposed to themagnetic field gradient along its length (in the y-direction) wasincreased to 12 mm as described above.

In FIG. 4B, the magnetic field and the magnetic field gradient arepresented as a function of distance from the collection stream flow pathchannel wall 297. The solid, dashed, and dotted lines in the graphs ofFIG. 4B correspond to the vertical magnetic field (B_(z)), horizontalmagnetic field (B_(x)) and the magnetic field gradient across thechannel

$\left( {\frac{\overset{\rightarrow}{B}}{x} \cdot \hat{B}} \right),$

respectively. In FIG. 4C, computer-simulated magnetic fielddistributions are depicted as grayscale variations within themicrofluidic channel generated by the magnetized high gradient magneticfield concentrator 290 in the form of a NiFe microcomb. Both top (topFIG. 4C) and cross-sectional (bottom FIG. 4C) views are illustrated.

To quantify the separation efficiency and to further analyze theperformance of the micromagnetic separator device with the NiFemicrocomb high gradient magnetic field concentrator for magneticparticle separation, green fluorescent magnetic beads (1.6 μm diameter;1.6×10⁷ beads/ml) were mixed with red fluorescent non-magnetic beads (2μm diameter; 2.2×10⁷ beads/ml) in PBS and introduced into the sourcepath as shown in FIG. 5A (top). FIGS. 5A-C were generated by overlayingsequential frames of corresponding movies taken at the beginning,middle, and end (left to right) of the microfluidic channel, in theabsence (top) or presence (bottom) of each pair of images.

Without magnetization, both the magnetic and non-magnetic beads followedtheir laminar source flow path and thus, both the red and greenmicrobeads exited from the top source outlet as shown in FIG. 5A (top).When the NiFe high gradient magnetic field concentrator microcomb wasmagnetized, almost all of the green magnetic beads observed under themicroscope were pulled from the source flow path 222 across the laminarstreamline boundary 220 and into the collection stream flow path 224 andexited through the lower collection outlet 280, whereas the rednon-magnetic beads remained in the original upper source flow path asshown in FIG. 5A (bottom).

As shown in Table 1 below, quantification of the separation efficiencyof the magnetic beads at the collection outlet revealed that, at avolume flow rate of 40 μl/hr, 92% of the magnetic beads exited from thecollection outlet, whereas less than 1% of the non-magnetic beads werepresent in this fraction. The same green magnetic beads (1.6×10⁷beads/ml) were then mixed in isotonic saline with red dye (Syto64)-stained human RBCs at a concentration similar to that in blood(2×10⁹ cells/ml), and injected into the top source inlet of themicrofluidic channel as shown in FIG. 5B (top). Again, the device of thepresent invention efficiently separated the magnetic beads from theflowing RBCs using the on-chip HGMC-microfluidic separator. Theseresults are shown in FIG. 5B (bottom versus top). Also shown in Table 1,at a flow rate of 25 μl/hr, 83% of the magnetic beads and less than 1%of RBCs were retrieved from the collection outlet. This also confirmedthat the effect of the magnetic force generated by the magnetized NiFelayer on RBCs is insignificant in this system.

With further regard to Table 1, the “flow rate” column is indicative ofthe flow rate of the source flow path in the microfluidic channel. Atleast 10 μl of fluid volume was collected for quantification using theflow rate indicated. “Throughput” of the system was estimated based onthe flow rate and the cell or bead density of the sample. The magneticnanoparticles used for labeling E. coli were not included whencalculating throughput. The separation efficiencies are depicted inFIGS. 5A-5C and were calculated in two ways. The left column of theseparation efficiency entry was calculated usingI_(c,mag)/(I_(c,mag)+I_(s,mag)). The right column of the separationefficiency entries was calculated using I_(c mag)/(I_(s,non-mag), whereI_(c mag) and I_(s,mag) are the intensity (fluorescence or OD_(600nm))of beads or cells collected at the lower outlet and upper outlet,respectively, with magnetic field turned on, and I_(s,non-mag) is theintensity (fluorescence or OD_(600nm)) of beads or cells collected atthe upper source outlet with the magnetic field turned off.Additionally, the amount of non-magnetic beads or RBCs collected at thelower collection outlet was less than 1% of the amount of non-magneticbeads or RBCs collected at the upper source outlet in all tests. Withregard to test number 3, for better visualization of the streamlineboundary, the PBS buffer contained Texas Red-conjugated bovine serumalbumin (0.1 mg/ml). Additionally, with regard to test number 4, themagnetic sample components included E. coli (5×10⁶ cells/ml)+130 nmmagnetic particles (5×10⁹ particles/ml). Similarly, in test 5, themagnetic sample components included E. coli (5×10⁶ cells/ml)+130 nmmagnetic particles (1.0×10¹⁰ particles/ml).

TABLE 1 Results of sorting particles and cells using the combinedmicrofluidic-micromagnetic separator with the NiFe microcomb FlowThroughput Sample components rate (beads or Separation Test MagneticNon-magnetic (μl/hr) cells/s) efficiency (%) 1. 1.6 μm beads 2 μm beadsin PBS 40 420 92 ± 4 86 ± 6 2. 1.6 μm beads RBCs in saline 25 10,000 83± 5 79 ± 5 3. E. coli + 130 nm beads PBS 30 80 89 ± 6 83 ± 9 4. E.coli + 130 nm beads RBCs in saline 25 10,000 53 ± 8  44 ± 11 5. E.coli + 130 nm beads RBCs in saline 25 10,000  78 ± 10 70 ± 9

In one embodiment of the present invention, living E. coli bacteria wereseparated by the microfluidic separation system. The separationtechniques were performed using the on-chip HGMC-microfluidic separatoron living E. coli bacteria in flowing fluids, both alone and when mixedwith RBCs. In these studies, 130 nm magnetic particles were used tolabel E. coli bacteria (1×10⁷ cells/ml) by incubating the cells withbiotinylated anti-E. coli antibody, mixing them with 130 nm magneticnanoparticles coated with streptavidin (1.0×10¹⁰ particles/ml) in PBS,and then injecting them into the source inlet of the microfluidicchannel. Nanometer-sized (130 nm) were used to label the bacteriabecause they bind more efficiently to E. coli compared tomicrometer-sized magnetic beads with similar surface functionality,likely due to the increased steric hindrance with micrometer-sizedbeads. Further, magnetic nanoparticles are less likely to occlude smallvessels and have longer circulation times than microbeads (Gupta, 2004)and are better suited for the in-line microfluidic separation device forcleansing blood or other biological fluids of biopathogens, such as inseptic patients.

Upon activating the magnetic field gradient, almost all of the observedE. coli cells originally confined to the upper laminar source flow pathas shown in FIG. 5C (top) were moved to the lower collection stream flowpath and passed out through the collection outlet as shown in FIG. 5C(bottom). At a flow rate of 30 μl/hr, 89% the E. coli cells wereseparated from the source original flow path. Similar studies confirmedthat E. coli (5×10⁶ cells/ml; 0.5×10¹⁰ magnetic nanoparticles/ml) couldbe separated from saline containing a physiological concentration ofRBCs (2×10⁹ cells/ml), but the separation efficiency of E. coli at thecollection outlet was 53% at a flow rate of 25 μl/hr. This decreasedseparation efficiency may be due to the increased viscosity of thisfluid which contains RBCs, as opposed to PBS. However, the separationefficiency was greatly improved when the ratio of magnetic nanoparticlesto bacteria was increased. For example, as shown in Table 1, at the sameflow rate, 78% of the E. coli bacteria were retrieved through thecollection outlet in a single pass when twice the amount of the magneticparticles were utilized (5×10⁶ cells/ml; 1.0×10¹⁰ magneticnanoparticles/ml).

The separation efficiency of the magnetic entities at the collectionoutlet of the microfabricated on-chip microfluidic-micromagnetic cellseparator device ranged from 78% to over 90% at volume flow rates of 25μl/hr to 40 μl/hr. As listed in Table 1, at low magnetic bead or celldensities on the order of approximately 10⁷ beads or E. coli/ml, athroughput of more than 80 beads or cells/s was routinely achieved usingthe micromagnetic separator device. Moreover, when sorting samples witha high cell density, such as approximately 10⁹ RBCs/ml, for example, thethroughput of the microdevice increased to 10,000 cells/s, as alsolisted in Table 1.

Both E. coli and the magnetic nanoparticles have multiple binding sitesavailable on their surfaces, and thus they are potential crosslinkersand upon mixing, can form large clusters composed of multiple E. colibacteria. Such clusters will have a much larger effective diameter thanan individual E. coli bacterium bound to magnetic particles and hence,they will exhibit a decreased magnetic deviation distance in thex-direction. See Equation (1) in the Appendix. Increasing the ratio ofmagnetic nanoparticles to bacteria reduces the formation of suchclusters. When the ratio of magnetic nanoparticles to bacteria wasdoubled, the separation efficiency of E. coli from the fluids containinga physiological concentration of RBCs increased from 53% to 78%. Thisincreased separation efficiency may be due to the reduction in both thesize and number of E. coli-magnetic nanoparticle clusters.

Heterogeneity in the size and magnetic properties of magneticsusceptible components in the source mixture results in a widedistribution of magnetic deviation distances in the x-direction duringcontinuous separation (see Appendix). Although this is beneficial forapplications such as on-chip magnetophoresis (Pamme and Manz, 2004), formagnetic separations of bacteria or cells from biological fluids,variations in magnetic deviation distance need to be minimized. In thesystem of the present invention, a viscous dextran solution was used asthe collection medium for this purpose, but other viscous solutions suchas albumin or lipid solutions, and the like may also be used. SeeEquation (4) in the Appendix. These viscous solutions serve as thecollection media to focus distribution of the magnetically susceptibletarget material in the collection stream flow path based upon a magneticdeviation distance. That is, the dextran and other viscous solutionsprovide a collection flow path that serves to focus the target materialwithin an acceptable range of distances where the material may be easilycollected via the collection outlet. With a suitable viscous solution,the magnetically susceptible materials will not overshoot the collectionstream flow path based on their strong attraction to the high gradientmagnetic field concentrator, nor will they undershoot the collectionstream flow path due to a collection media that provides too muchresistance for the magnetically susceptible particles to overcome. Inthe system of the present invention, even though occasional sampletrapping on the collection side of the channel wall was observed, thiseffect was small, as indicated by the less than a 10% difference betweenthe separation efficiencies of the magnetic beads and cells calculatedwith two methods as shown in Table 1 and described above.

In the system of the present invention, the high gradient magnetic fieldconcentrator, such as the NiFe layer, for example, is positioned outsidethe microfluidic channel to eliminate concerns for the biocompatabilityof the magnetic materials used. For example, the nickel used in pastmagnetic separation applications (Han and Frazier, 2004, 2006) mayexhibit biocompatibility problems (Takamura et al., 1994; Uo et al.,1999; Wataha et al., 2001). Also, by placing the high gradient magneticfield concentrator at a distance (100 μm) from the channel, magneticparticles are less likely to be trapped by the magnetic field at thechannel edge. The magnetic field gradient created is effective atdriving movement of magnetic microbeads or E. coli labeled with magneticnanoparticles into the collection stream flow path while notsignificantly displacing RBCs that may be slightly magnetic because theycontain deoxyhemoglobin (Melville et al., 1975b; Takayasu et al., 1982)(see Appendix).

The foregoing description of exemplary aspects and embodiments of thepresent invention provides illustration and description, but is notintended to be exhaustive or to limit the invention to the precise formdisclosed. Those of skill in the art will recognize certainmodifications, permutations, additions, and combinations of thoseembodiments are possible in light of the above teachings or may beacquired from practice of the invention. Therefore, the presentinvention also covers various modifications and equivalent arrangementsthat would fall within the purview of appended claims and claimshereafter introduced.

APPENDIX High Gradient Magnetic Field Concentrator-Microfluidic DesignAnalysis & Development Design Analysis

The force on a magnetic particle aligned with a magnetic field is givenby {right arrow over (F)}_(mag)=m{circumflex over (B)}·∇{right arrowover (B)}, where m is the magnetic moment of the particle, B is themagnetic field, and {right arrow over (B)} is the unit vector in thedirection of B. In the microfluidic channel with fluid flow in they-direction and a perpendicular magnetic field gradient in thex-direction, material such as magnetic particles in the fluid will shifttoward the maximum of the magnetic field, cross he streamline bondary,and traverse the microfluidic channel in the x-direction. See FIG. 2inset. After passing through the magnetic field, the particle's finaldistance from the source flow side of channel wall, that is the upperchannel edge 299 in FIG. 2 inset, X_(final) is approximated by:

$\begin{matrix}{X_{final} = {\frac{\left( {m{\frac{\overset{\rightarrow}{B}}{x} \cdot \hat{B}}} \right)L_{y}}{3\; \pi \; \eta \; {Dv}_{y}} + X_{initial}}} & (1)\end{matrix}$

Assuming that (1) the magnetic field gradient is constant across thewidth of the channel in the x-direction, (2) the magnetic field isconstant across the height of the channel in the z-direction, (3) themagnetic force in the y-direction is much smaller than the Stokes dragon the particle, and (4) the source flow and collection flow havesimilar fluid viscosity η. In Equation (1), X_(initial) is the distanceof the particle from the source flow side of channel wall beforeentering the magnetic field, D is the particle's effective diameter,L_(y) is the span of the magnetic field in the y-direction, and v_(y) isthe particle's flow velocity in the y-direction.

For maximum cleansing of the biological fluid, it is crucial to maximizethe separation efficiency of magnetic particles, that is, the percentageof magnetic particles that are moved from the source flow path into thecollection stream flow path during passage through the HGMC of themicrofluidic channel. On the other hand, it is also very important tominimize the loss of the non-magnetic particles from the source flowpath, that is, to minimize the percentage of non-magnetic particles thatmove into the collection stream flow path during the separation process.In the present invention, there are two possible causes for this loss,namely diffusion and the native magnetic susceptibility of a few celltypes, for example, RBCs containing deoxyhemoglobin.

Diffusion in the system of the present invention is determined byd=√{square root over (D₁L/ v)}, where D₁ is the diffusion coefficient, dis the diffusion distance, L is the channel length, and v is the averageflow rate. Diffusion is undesirable for the embodiments described abovedue to the possible loss of critical biomolecules or cells frombiological fluids, for example, blood proteins, platelets, and the like.The diffusion coefficients of the smaller proteins are on the order of10 μm²/s in water, and they are even smaller in more viscous media.Assuming D₁=30 μm²/s and the acceptable diffusion distance as 10% of thechannel width, the determined maximum time that a fluid volume elementshould be in the channel is

${{L/\overset{\_}{v}} \leq {3.3 \times 10^{8}\mspace{14mu} {\frac{s}{m^{2}} \cdot W^{2}}}},$

in which W is channel width. Furthermore, by setting L_(y)=kL (0≦k≦1)and v_(y)≈ v, Equation (1) is converted to:

$\begin{matrix}{X_{final} \leq {\frac{3.3 \times 10^{8}{k\left( {m{\frac{\overset{\rightarrow}{B}}{x} \cdot \hat{B}}} \right)}W^{2}}{3\; \pi \; \eta \; D}\mspace{14mu} m}} & (2)\end{matrix}$

RBCs containing deoxyhemoglobin reportedly have a relative magneticsusceptibility in water (or plasma) of about 3.9×10⁻⁶. (Melville et al.,1975b; Takayasu et al., 1982). To prevent the loss of RBCs from thesource flow path in the system of the present invention, the acceptabledeviation of deoxyhemoglobin RBCs in the x-direction after passingthrough the magnetic field (X_(final,RBC)−X_(initial,RBC)) was set to1/100 of the channel width, that is, W/100.

Design Development

For a magnetic particle at given flow conditions, Equation (1) indicatesthat X_(final) is a function of m and

$\frac{\overset{\rightarrow}{B}}{x} \cdot {\hat{B}.}$

When a magnetic particle is unsaturated {right arrow over (m)}=χV{rightarrow over (B)}/μ₀, where χ is the magnetic permeability of theparticle, V is the volume of the particle, and μ₀ is the magneticpermeability of vacuum. As B increases, m approaches a saturation valuem_(s). For convenience, the value of m_(s)μ₀/χV is referred to as thesaturation magnetic field of the particle B_(s).

The majority of bioorganisms are non-magnetic, and need to be labeledwith superparamagnetic particles in order to be separated from thesource mixture. In the system of the present invention, thesuperparamagnetic particles used to label E. coli are 130 nm indiameter, and have a magnetic permeability χ_(bead)=12 with B_(s) of0.02 T. Assuming η=10 ⁻³ Pa·s (water at 20° C.), D=3×10⁻⁶ m (E. coli)and B>B_(s), it was inferred from Equation (2) that to separate E. colibound to a number n of the superparamagnetic particles from the sourcemixture,

${\frac{\overset{\rightarrow}{B}}{x} \cdot \hat{B}} > {{0.2/{knW}}\mspace{11mu} {T/{m.}}}$

Based on their size, an E. coli cell surface is estimated to be able toaccommodate over 800 of such superparamagnetic particles. If the cut-offvalue for n is set to 40, that is the system of the present inventionneeds to remove E. coli bound to at least 40 superparamagnetic particlesfrom the source mixture,

$\frac{\overset{\rightarrow}{B}}{x} \cdot \hat{B}$

should be at least 5.0×10⁻³/kWT/m.

In the system of the present invention, B and

$\frac{\overset{\rightarrow}{B}}{x} \cdot \hat{B}$

inside the channel are determined by the magnetic properties, geometry,and position of the high gradient magnetic field concentrator magneticlayer and the external magnetic field. Multiple types of magneticmaterials may be used to fabricate the magnetic layer. In one embodimentof the present invention, a soft magnetic material (NiFe) with lowremnant magnetization was chosen that was magnetized with an externalstationary magnet to facilitate rapid and switchable control ofseparations. The NiFe layer in one embodiment of the present inventionhas a saturation magnetization ˜0.6 T (Rasmussen, 2001). Other magneticmaterials may be utilized as the high gradient magnetic fieldconcentrator including paramagnetic materials, permanent magneticmaterials, electromagnetic devices, and the like.

Two NiFe layer geometries were selected in the embodiments of thepresent invention namely, a microneedle as detailed in FIG. 3 and amicrocomb as detailed in FIG. 4. The microneedle geometry concentratedthe magnetic field at one position along the channel and served as aproof of principle for our fabrication technology and manipulationstrategy. The microcomb geometry provided a field gradient along alonger stretch of the microfluidic channel, exposing magnetic particlesto force for a longer duration. The magnetic field and field gradientgenerated by the two NiFe layer geometries were determined by finiteelement simulations with Maxwell 3D (Ansoft), which solved for magneticfield on a mesh of tetrahedrons that matched the actual device geometryand included the B-H curve of the NiFe layer and the permanent magnet asshown in FIG. 3 and FIG. 4.

In an embodiment of the separation device of the present invention, theNiFe layer was positioned outside the microfluidic channel to eliminateconcerns for the biocompatability of the magnetic materials used. FIG. 4indicates that both B and

$\frac{\overset{\rightarrow}{B}}{x} \cdot \hat{B}$

depend on the distance between the magnetic layer and the microfluidicchannel. It was determined previously that

$\frac{\overset{\rightarrow}{B}}{x} \cdot \hat{B}$

should be at least 5.0×10⁻³/kWT/m, in which k corresponds to the ratiobetween the span of the magnetic layer in the y-direction L_(y) and themicrofluidic channel length L. k was set as 0.6 for the microcomb typeof magnetic layer to ensure accuracy in device assembly, and themicrofluidic channel width W was set as 200 μm. Since

$\frac{\overset{\rightarrow}{B}}{x} \cdot \hat{B}$

needs to be larger than 5.0×10⁻³/kW=42 T/m, the distance between thelayer edge and the collection flow side of channel wall was set to 100μm. Based on FIG. 4,

$\frac{\overset{\rightarrow}{B}}{x} \cdot \hat{B}$

inside the channel is approximately 55-250 T/m with 0.017 T<B_(z)<0.02 Tand 0.024 T<B_(x)<0.048 T. It was further calculated that assumingD_(RBC)=7 μm, and χ_(RBC)=3.9×10⁻⁶ (X_(final,RBC)−X_(initial,RBC)) isless than 0.1 μm, and less than 1/100 of the channel width, confirmingthat the loss of RBCs from source flow after passing through themagnetic field is negligible.

Finally, for magnetic separations of E. coli and other bioorganisms orcells as well, the magnetic susceptible entities in the source mixtureinclude both E. coli bound to a wide range number of superparamangeticparticles and the superparamagnetic particles themselves. Thisheterogeneity leads to large variations in X_(final). To minimize suchvariations as (X_(final,max)−X_(final,min))/W, a media with higher fluidviscosity η_(c) was used in the collection stream flow path than in thesource flow path (η_(s)). By setting η_(c)=pη_(s)(p>1), Equation (1) isre-written as:

$\begin{matrix}{X_{final} = \left\{ \begin{matrix}{{\frac{\left( {m{\hat{B} \cdot {\nabla\overset{\rightarrow}{B}}}} \right)L_{y}}{3\; \pi \; \eta \; {Dv}_{y}} + X_{inital}},} & {{{{when}\mspace{14mu} \frac{\left( {m{\hat{B} \cdot {\nabla\overset{\rightarrow}{B}}}} \right)L_{y}}{3\; \pi \; \eta \; {Dv}_{y}}} + X_{inital}} \leq \frac{W}{2}} \\{\begin{matrix}{\frac{\left( {m{\hat{B} \cdot {\nabla\overset{\rightarrow}{B}}}} \right)L_{y}}{3\; \pi \; \eta \; {pDv}_{y}} + \frac{X_{inital}}{p} +} \\\frac{\left( {p - 1} \right)W}{2\; p}\end{matrix},} & {{{{when}\mspace{14mu} \frac{\left( {m{\hat{B} \cdot {\nabla\overset{\rightarrow}{B}}}} \right)L_{y}}{3\; \pi \; \eta \; {Dv}_{y}}} + X_{inital}} > \frac{W}{2}}\end{matrix} \right.} & (3)\end{matrix}$

Comparing the variations in X_(final) when media with fluid viscosity ofη_(s), and η_(c) are used in the collection stream flow pathrespectively, and when both X_(final,max) and X_(final,min) are largerthan W/2, Equation (3) gives:

$\begin{matrix}{\frac{\left( {X_{{final},\max} - X_{{final},\; \min}} \right)_{\eta_{c}}}{\left( {X_{{final},\max} - X_{{final},\; \min}} \right)_{\eta_{s}}} = {\frac{1}{p} < 1}} & (4)\end{matrix}$

Hence, using more viscous media in the collection stream flow path canreduce the variations in X_(final). In one embodiment of the presentinvention, a dextran solution, which is both viscous and biocompatible,was used as the fluid medium for the collection stream flow path.

1. An integrated microfluidic separator device comprising: amicrofluidic channel including: a source flow path including a sourceinlet and a source outlet, and a collection stream flow path including acollection outlet, wherein a laminar streamline boundary separates thesource flow path and the collection stream flow path; a microfabricatedhigh-gradient magnetic concentrator with a high curvature geometryintegrated at a first side of the microfluidic channel; and a magneticsource to magnetize the microfabricated high gradient magnetic fieldconcentrator to move magnetically susceptible target material flowing inthe source flow path to cross over the laminar streamline boundary andenter the collection stream flow path to separate the magneticallysusceptible target material from a fluid flowing in the microfluidicchannel.
 2. The microfluidic separator device of claim 1, wherein thecollection stream flow path includes a viscous dextran solution as acollection media to focus distribution of the magnetically susceptibletarget material in the collection medium based upon a magnetic deviationdistance.
 3. The microfluidic separator device of claim 1, wherein thecollection stream flow path includes a viscous solution comprising atleast one of albumin or lipid solutions as a collection media to focusdistribution of the magnetically susceptible target material in thecollection medium based upon a magnetic deviation distance.
 4. Themicrofluidic separator device of claim 2, wherein the high curvaturegeometry of the microfabricated high-gradient magnetic concentrator is amicroneedle.
 5. The microfluidic separator device of claim 2, whereinthe high curvature geometry of the microfabricated high-gradientmagnetic concentrator is a microcomb.
 6. The microfluidic separatordevice of claim 5, wherein the microfabricated high-gradient magneticconcentrator microcomb includes a triangular saw-tooth configuration. 7.The microfluidic separator device of claim 2, wherein the high curvaturegeometry of the microfabricated high-gradient magnetic fieldconcentrator includes a magnetic line array.
 8. The microfluidicseparator device of claim 2, wherein the microfabricated high-gradientmagnetic field concentrator comprises permanent magnetic materials thatdo not require an external magnetizing field.
 9. The microfluidicseparator device of claim 2, wherein the magnetically susceptible targetmaterial is labeled with at least one of paramagnetic particles orsuper-paramagnetic particles to facilitate separation of the targetmaterial from the fluid.
 10. The microfluidic separator device of claim9, wherein the at least one of paramagnetic particles orsuper-paramagnetic particles bind to the magnetically susceptible targetmaterial to facilitate separation of the magnetically susceptible targetmaterial from a biological fluid.
 11. The microfluidic separator deviceof claim 10, wherein the separated magnetically susceptible targetmaterial includes at least one of microbial or mammalian cells.
 12. Themicrofluidic separator device of claim 10, wherein the separatedmagnetically susceptible target material includes at least one ofmolecules or chemicals.
 13. The microfluidic separator device of claim10, wherein the separated magnetically susceptible target materialincludes at least one of bacterial, viral, or fungal pathogens.
 14. Themicrofluidic separator device of claim 10, wherein the separatedmagnetically susceptible target material includes at least one of tumorcells or stem cells.
 15. The microfluidic separator device of claim 10,wherein the at least one of paramagnetic particles or super-paramagneticparticles are coated with biomolecules and bind to components of thesurface of the target material in the fluid.
 16. The microfluidicseparator device of claim 15, wherein the biomolecules are at least oneof proteins, nucleotides, carbohydrates, or lipids.
 17. The microfluidicseparator device of claim 15, wherein the biomolecules are antibodymolecules.
 18. The microfluidic separator device of claim 17, whereinthe coating of the at least one of paramagnetic particles orsuper-paramagnetic particles coated with antibody molecules is performedby covalent cross-linking techniques.
 19. The microfluidic separatordevice of claim 17, wherein the biomolecules include natural opsonins.20. The microfluidic separator device of claim 2, wherein themicrofluidic channel and the microfabricated high-gradient magneticconcentrator are microfabricated on a single chip.
 21. The microfluidicseparator device of claim 20, wherein the magnetic source ismicrofabricated on the single chip.
 22. The microfluidic separatordevice of claim 20, wherein the magnetic source is at least one of apermanent magnet, a paramagnetic source, or an electromagnet and isexternal to the single chip.
 23. The microfluidic separator device ofclaim 20, wherein the microfabricated high-gradient magneticconcentrator comprises permanent magnetic materials that do not requirean external magnetizing field.
 24. The microfluidic separator device ofclaim 2, wherein the microfabricated high-gradient magnetic concentratorincludes a layer of magnetizable material.
 25. The microfluidicseparator device of claim 24, wherein the microfabricated high-gradientmagnetic concentrator is magnetized by a paramagnetic magnetic source.26. The microfluidic separator device of claim 24, wherein the layer ofmagnetizable material is magnetized using a magnetic source including anelectromagnet.
 27. The microfluidic separator device of claim 24,wherein the layer of magnetizable material is magnetized using amagnetic source including a permanent magnet.
 28. The microfluidicseparator device of claim 27, wherein the permanent magnet comprisesneodymium.
 29. The microfluidic separator device of claim 24, whereinthe microfabricated high-gradient magnetic concentrator is positionedoutside the microfluidic channel.
 30. The microfluidic separator deviceof claim 24, wherein the microfabricated high-gradient magneticconcentrator is positioned within the microfluidic channel.
 31. Themicrofluidic separator device of claim 29, wherein the microfabricatedhigh-gradient magnetic concentrator positioned outside the microfluidicchannel includes a layer of NiFe.
 32. The microfluidic separator deviceof claim 30, wherein the microfabricated high-gradient magneticconcentrator positioned within the microfluidic channel includes a layerof NiFe.
 33. The microfluidic separator device of claim 29, wherein thelayer of magnetizable material of the microfabricated high-gradientmagnetic concentrator is substantially the same thickness as the heightof the microfluidic channel to subject the channel to a substantiallyuniform magnetic field gradient.
 34. The microfluidic separator deviceof claim 24, wherein the position of the microfabricated high-gradientmagnetic concentrator and the laminar fluid flow prevents accumulationof collected target material at sidewalls of the channel and therebyprovides continuous fluid flow in the microfluidic channel.
 35. Themicrofluidic separator device of claim 2, wherein the magneticallysusceptible target material that crosses over the laminar streamlineboundary and enters the collection stream flow path exits themicrofluidic channel through the collection outlet.
 36. The microfluidicseparator device of claim 2, wherein material flowing in the source flowpath that is not magnetically susceptible exit the microfluidic channelthrough the source outlet.
 37. The microfluidic separator device ofclaim 2, wherein the microfabricated high-gradient magnetic concentratorprovides a horizontal magnetic field of at least 0.025 T and a verticalmagnetic field of at least 0.018 T and a field gradient of at least 50T/m.
 38. The microfluidic separator device of claim 2, wherein theseparated target material includes microbial cells.
 39. The microfluidicseparator device of claim 38, wherein the microbial cells are E. colibacteria.
 40. The microfluidic separator device of claim 38, wherein themicrobial cells are Candida Albicans fungi.
 41. The microfluidicseparator device of claim 2, wherein the separated target materialincludes mammalian cells.
 42. The microfluidic separator device of claim41, wherein the mammalian cells include tumor cells.
 43. Themicrofluidic separator device of claim 41, wherein the mammalian cellsinclude stem cells.
 44. The microfluidic separator device of claim 41,wherein the mammalian cells include blood cells.
 45. The microfluidicseparator device of claim 38, wherein the microbial cells are labeledwith at least one of biocompatible paramagnetic particles orbiocompatible super-paramagnetic particles to facilitate separation ofthe microbial cells from a biological fluid.
 46. The microfluidicseparator device of claim 41, wherein the mammalian cells are labeledwith at least one of biocompatible paramagnetic particles orbiocompatible super-paramagnetic particles to facilitate separation ofthe mammalian cells from a biological fluid.
 47. The microfluidicseparator device of claim 46, wherein the biological fluid includes atleast one of blood, serum, urine, cerebrospinal fluid, tracheaaspirates, saliva, tears, or perspiration.
 48. A method of separatingtarget material from a fluid in continuous laminar flow, the methodcomprising: introducing the fluid containing a magnetically labeledtarget material through a source inlet into a source flow path of amicrofluidic channel, wherein the microfluidic channel includes a sourceflow path with a source inlet and a source outlet and a collectionstream flow path with a collection outlet, and wherein a laminarstreamline boundary separates the source flow path and the collectionstream flow path; and magnetizing a microfabricated high-gradientmagnetic field concentrator with a local magnetic field gradient to movethe labeled target material from the source flow path into thecollection stream flow path to a collection outlet to selectively removethe target material from the fluid in continuous laminar flow, whereinthe microfabricated high-gradient magnetic field concentrator includes ahigh curvature geometry and the microfabricated high-gradient magneticfield concentrator is adjacent to a first side of the microfluidicchannel.
 49. The method of separating target material of claim 48,wherein the collection stream flow path includes a viscous dextransolution as a collection media to focus distribution of the magneticallysusceptible target material in the collection medium based upon amagnetic deviation distance.
 50. The method of separating targetmaterial of claim 48, wherein the collection stream flow path includes aviscous solution comprising at least one of albumin or lipid solutionsas a collection media to focus distribution of the magneticallysusceptible target material in the collection medium based upon amagnetic deviation distance.
 51. The method of separating targetmaterial of claim 49, further comprising: positioning a magnetic sourcesubstantially in the middle of the microfabricated high-gradientmagnetic field concentrator to magnetize the microfabricatedhigh-gradient magnetic field concentrator with a local magnetic fieldgradient.
 52. The method of separating target material of claim 49,wherein the high curvature geometry of the microfabricated high-gradientmagnetic field concentrator is a microneedle.
 53. The method ofseparating target material of claim 49, wherein the high curvaturegeometry of the microfabricated high-gradient magnetic fieldconcentrator is a microcomb.
 54. The method of separating targetmaterial of claim 53, wherein the microfabricated high-gradient magneticconcentrator microcomb includes a triangular saw-tooth configuration.55. The method of separating target material of claim 49, wherein thehigh curvature geometry of the microfabricated high-gradient magneticfield concentrator includes a magnetic line array.
 56. The method ofseparating target material of claim 49, wherein the microfabricatedhigh-gradient magnetic field concentrator comprises permanent magneticmaterials that do not require an external magnetizing field.
 57. Themethod of separating target material of claim 49, wherein the labeledtarget material includes magnetically susceptible cells labeled with atleast one of biocompatible paramagnetic particles or biocompatiblesuper-paramagnetic particles to facilitate separation of themagnetically susceptible cells from a biological fluid.
 58. The methodof separating target material of claim 57, wherein the separatedmagnetically susceptible target material includes at least one ofmicrobial or mammalian cells.
 59. The method of separating targetmaterial of claim 57, wherein the separated magnetically susceptibletarget material includes at least one of molecules or chemicals.
 60. Themethod of separating target material of claim 57, wherein the separatedmagnetically susceptible target material includes at least one ofbacterial, viral, or fungal pathogens.
 61. The method of separatingtarget material of claim 57, wherein the separated magneticallysusceptible target material includes at least one of tumor cells or stemcells.
 62. The method of separating target material of claim 57, whereinthe at least one of paramagnetic particles or super-paramagneticparticles are coated with biomolecules and bind to components on asurface of the target material in the fluid.
 63. The method ofseparating target material of claim 62, wherein the biomolecules are atleast one of proteins, nucleotides, carbohydrates, or lipids.
 64. Themethod of separating target material of claim 62, wherein the at leastone of paramagnetic particles or super-paramagnetic particles are coatedwith antibody molecules.
 65. The method of separating target material ofclaim 64, wherein the coating of the at least one of paramagneticparticles or super-paramagnetic particles coated with antibody moleculesis performed by covalent cross-linking techniques.
 66. The method ofseparating target material of claim 62, wherein the at least one ofparamagnetic particles or super-paramagnetic particles are coated withnatural opsonins.
 67. The method of separating target material of claim49, wherein the microfabricated high-gradient magnetic concentratoradjacent to a first side of the microfluidic channel is positionedoutside the microfluidic channel.
 68. The method of separating targetmaterial of claim 49, wherein the microfabricated high-gradient magneticconcentrator adjacent to a first side of the microfluidic channel ispositioned within the microfluidic channel.
 69. The method of separatingtarget material of claim 49, wherein the position of the microfabricatedhigh-gradient magnetic concentrator and the laminar fluid flow preventsaccumulation of collected target material at sidewalls of the channeland thereby provides continuous fluid flow in the microfluidic channel.70. The method of separating target material of claim 51, furthercomprising microfabricating the microfluidic channel and themicrofabricated high-gradient magnetic field concentrator on a singlechip.
 71. The method of separating target material of claim 70, furthercomprising microfabricating the magnetic source on the single chip. 72.The method of separating target material of claim 70, wherein themagnetic source is at least one of a permanent magnet, a paramagneticsource, or an electromagnet and is external to the single chip.
 73. Themethod of separating target material of claim 71, wherein themicrofabricated high-gradient magnetic concentrator comprises permanentmagnetic materials that do not require an external magnetizing field.74. The method of separating target material of claim 51, wherein themicrofabricated high-gradient magnetic concentrator includes a layer ofmagnetizable material.
 75. The method of separating target material ofclaim 74, wherein the layer of magnetizable material includes NiFe. 76.The method of separating target material of claim 75, wherein the layerof NiFe is magnetized using the magnetic source including a permanentmagnet.
 77. The method of separating target material of claim 75,wherein the layer of NiFe is magnetized using the magnetic sourceincluding an electromagnet.
 78. The method of separating target materialof claim 75, wherein the layer of NiFe is magnetized by a paramagneticmagnetic source.
 79. The method of separating target material of claim76, wherein the permanent magnet comprises neodymium.
 80. The method ofseparating target material of claim 70, wherein a plurality ofintegrated microfluidic separator devices are included at a plurality ofpositions on the single chip to provide multiplexing separation oftarget material from the fluid and wherein each microfluidic separatordevice includes at least one high gradient magnetic field concentrator.81. The method of separating target material of claim 49, furthercomprising moving target material in the continuous flow that isunaffected by the magnetization of the microfabricated high-gradientmagnetic field concentrator through the source flow path to a sourceoutlet.
 82. The method of separating target material of claim 49,wherein the separated target material includes microbial cells.
 83. Themethod of separating target material of claim 82, wherein the microbialcells include E. coli bacteria.
 84. The method of separating targetmaterial of claim 82, wherein the microbial cells include CandidaAlbicans fungi.
 85. The method of separating target material of claim49, wherein the target material includes mammalian cells.
 86. The methodof separating target material of claim 85, wherein the mammalian cellsinclude tumor cells.
 87. The method of separating target material ofclaim 85, wherein the mammalian cells include stem cells.
 88. The methodof separating target material of claim 85, wherein the mammalian cellsinclude blood cells.
 89. The method of separating target material ofclaim 82, wherein the microbial cells are labeled with at least one ofbiocompatible paramagnetic particles or biocompatible super-paramagneticparticles to facilitate separation of the microbial cells from abiological fluid.
 90. The method of separating target material of claim85, wherein the mammalian cells are labeled with at least one ofbiocompatible paramagnetic particles or biocompatible super-paramagneticparticles to facilitate separation of the mammalian cells from abiological fluid.
 91. The method of separating target material of claim90, wherein the biological fluid includes at least one of blood, serum,urine, cerebrospinal fluid, trachea aspirates, saliva, tears, orperspiration.
 92. A microfluidic separator system comprising a pluralityof integrated microfluidic separator devices included at a plurality ofpositions on a single chip to provide multiplexing separation of targetmaterial from a fluid, and wherein each microfluidic separator deviceincludes: a microfluidic channel including: a source flow path includinga source inlet and a source outlet, and a collection stream flow pathincluding a collection outlet, wherein a laminar streamline boundaryseparates the source flow path and the collection stream flow path; amicrofabricated high-gradient magnetic concentrator with a highcurvature geometry integrated at a first side of the microfluidicchannel; and a magnetic source to magnetize the microfabricated highgradient magnetic field concentrator to move magnetically susceptibletarget material flowing in the source flow path to cross over thelaminar streamline boundary and enter the collection stream flow path toseparate the magnetically susceptible target material from a fluidflowing in the microfluidic channel.